Focused ion beam scanning electron microscopy (FIB-SEM), also referred to as ion abrasion scanning electron microscopy (IA-SEM), is a technology that we have been developing in the lab to image cells and tissues in 3D at high resolution. Imaging cells and tissues by FIB-SEM at high resolution offers many exciting possibilities for biological research; however, at high resolution, this technology produces enormous amounts of data, and is extremely slow. Moreover, one of the most promising aspects of this technology is the ability to quantitatively analyze ultrastructural morphology. Thus in addition to using FIB-SEM to study 3D architecture in cells and tissues, we have also been developing imaging methods and techniques that align the technology with the goal of automated, quantitative analysis of 3D structure at electron microscopy resolutions. Over the last few years, we have developed a number of techniques to streamline FIB-SEM imaging. These have included novel tracking mechanisms to normalize and verify alignment and 3D reconstruction, as well as techniques to allow high resolution imaging of areas of interest without requiring extremely slow and data-intensive high resolution imaging of the entire volume. We are currently building on this technology with a variety techniques for identifying specific proteins or objects of interest. One such technique is correlative light and electron microscopy, where one registers a light fluorescent volume against the final 3D FIB-SEM volume, allowing one to narrow in on a specific area in the volume in order to identify an object of interest. Other new techniques that we are developing for FIB-SEM are clonable electron-dense tags, which allow direct visualization and identification of a tagged protein within the 3D volume. These tags have been used successfully in conventional 2D imaging; their use in 3D FIB-SEM imaging offers tremendous possibilities for being able to analyze protein localization in the context of cellular ultrastructure. Analysis of morphological cellular features by electron microscopy has long been a qualitative process, especially in light of cellular heterogeneity and the technical difficulties of obtaining sufficient sampling by this method. Building upon our work in 3D cellular analysis by FIB-SEM, we have undertaken a semi-quantitative study of morphological changes in cellular components during cell differentiation. Using C2C12 cells as a model system, we have applied a number of different analytical tools, including thresholding tools to identify subnuclear structures such as nuclear pore complexes, heterochromatin, euchromatin, and nucleoli, and shape tools to identify changes in size, dimension, and shape of the subcellular structures. We were able to quantitatively detect changes in organellar shape and composition during differentiation. These tools offer novel mechanisms for quantitative 3D ultrastructural analysis that are not dependent on fluorescence microscopy, and open the door for quantitative analysis of fine structures not visible even by super resolution light microscopy. While developing these techniques, we have also applied the FIB-SEM technology to new biological problems. One such project was a study of the biogenesis of vaccinia virions; the process of formation of these virions makes them a tempting vehicle for delivery of therapeutics for a variety of disorders, including other viruses such as HIV and influenza. Early in the virion formation process, crescents of lipid bilayer appear near the endoplasmic reticulum of infected cells; later, these crescents close into spherical immature virions, before maturing into a final brick shape. Earlier studies with electron tomography and/or serial sectioning were hampered by the difficulty of both imaging the forming virions at high resolution and imaging virions and cell in 3D, leaving little idea about the 3D shape of the crescent shapes and how these transition into closed virions. Our FIB-SEM analysis revealed that crescents form as partial spheres; when the spheres reach a certain size, the nucleoid is recruited to the forming virion. These crescents appear to share lipid bilayer with neighboring crescents, and occasionally with the endoplasmic reticulum as well. As the nucleoid condenses within the forming crescent, the lipid bilayer then closes around the nucleoid into a sphere, eventually detaching from neighboring virions and maturing into the smaller, brick-shaped virion. Another exciting area of FIB-SEM analysis has been a collaboration with the Balaban laboratory in NHLBI to visualize the 3D mitochondrial network in muscle cells. During peak contraction, the energy requirements for skeletal muscle can be intense, requiring significant transfer of energy from the area immediately surrounding blood vessels to the interior of the muscle fiber. It has been hypothesized that this energy transfer occurs via transfer of metabolites such as ATP; however, the distances involved indicate that this would require facilitated diffusion, which genetic evidence suggests is not required in muscles except during peak performance. Our 3D study of the mitochondria in skeletal muscle shows that the mitochondrial reticulum network extends directly and contiguously from the area surrounding the blood vessel deep into the muscle fiber, enabling direct energy transfer via electrical conduction within the mitochondria. While facilitated metabolite diffusion is likely important for peak performance, the electrical conduction through the mitochondrial reticulum, as demonstrated in this study, may provide the majority of skeletal muscle energy requirements.